Targeting neurons and photons for optogenetics
Nature Neuroscience 16, 805–815 (July 2013)
dream experiments will become possible with the application of these new approaches
Caenorhabditis elegans has 302 neurons, and the morphology of every neuron is known.
mammalian retina: the functional unit is often considered to be a mosaic of cells with the same properties, referred to as cell type.
“cell type” refers to a population of neurons that cannot practically be divided into smaller units
“cell class” refers to a population of neurons that is defined by some common property but which can be further divided into smaller populations.
a single retinal ganglion cell mosaic
Targeting the right neurons is still a largely unsolved problem, especially in species, such as non-human primates, where genetic manipulations are often not feasible.
approaches for targeting optogenetic probes, focusing on using viruses, alone or in combination with transgenics.
the virus used most frequently for targeting, the adeno-associated virus (AAV), has a coat protein that exists in 100 different variants in nature … changing the entry site from axons to soma or dendrites.
The thousands of viruses made by nature and the many variants made by researchers can therefore be thought of as a ‘Legoland’ for neuroscientists performing optogenetics experiments
the way viruses are made is highly modular: the different properties are stored in different plasmids, and by mixing these plasmids and adding them to cells the virus is self-assembled.
Replication-competent viruses are toxic to varying degrees
the genetic identity of these neurons (for example, expression of parvalbumin)
Virus targeting based on genetic identity.
The morphology and function of different cell types is to a large extent defined by the pattern of genes they express.
Past work has used the fact that some classes of neurons uniquely express particular signature genes—for instance, a large class of fast-spiking interneurons expresses parvalbumin
molecular tools, such as site-specific recombinases (for example, Cre or Flp) can be used to drive the expression of optogenetic probes from viruses infecting these cells.
Such conditional viruses can be made from DNA viruses, such as AAV or herpesviruses
The main drawback of the conditional virus approach is that it requires expression of a site-specific recombinase, typically using a transgenic animal.
The generation of a transgenic animal for a target neuronal type is both time consuming and unpredictable
Rabies– and herpesvirus-based retrograde labeling methods, while suitable for short-term studies over days, are too toxic for studies in which long-term expression is needed.
AAVs are excellent tools for anterograde delivery; however, existing AAVs are not exclusively anterograde, and further development of nontoxic, exclusively anterograde vectors is needed.
single-cell electroporation of a postsynaptic neuron and the subsequent initiation of a retrograde virus from only the electroporated neuron.
high-level, long-term expression has been shown to cause abnormal axonal morphology
This involves using two-photon microscopy to target a plasmid-filled patch pipette to individual neurons in vivo, followed by electroporation to deliver the plasmid to the cell under visual control.
Neurons can be targeted in this way on the basis of
- their somatodendritic morphology (using ‘shadowimaging’30),
- their genetic identity (using GFP expression as a marker) or
- their functional properties (such as tuned responses to sensory stimuli) for subsequent optogenetic activation.
Optogenetics: controlling cell function with light
Nature Methods 8, 24–25 (2011)